4A, B, C, D Timeline

1. 4A, B, C, D Timeline • Monday, October 1st • DNA & RNA Lecture • Tuesday, October 2nd • Pre-Lab Quiz, Lab 4A (if time) • Thursday, October 4th • Lab 4B (50 minutes) • Monday, October 8th • MAGIC MOUNTAIN! • Tuesday, October 9th • Step 12 from Lab B, Lab 4C • Thursday, October 11th • Lab 4D (demonstration), Post-Lab Quiz

2. 4A, B, C, D Prep. 4A Check supplies: sodium chloride, TRIS, EDTA, pH paper, HCl & NaOH 4B Check supplies: salmon sperm DNA, Get ice 4C Check supplies: salmon sperm DNA, gelatin, DPA, sulfuric acid, acetic acid, acetaldehyde, NaOH 10%, Cupric Sulfate 5% Prepare NaOH 10% and Cupric Sulfate 4D Dilute ethidium bromide

3. Lab 4A Making Solutions for DNA Isolation

4. Overview • Make 10 mL of 5 M NaCl solution • Make 100 mL of TE buffer • Keep buffer at 4 degrees Celsius until ready for use • 1 mM TRIS • Maintains pH of the DNA sample • 1 mM EDTA • Denatures Dnases and keeps the DNA sample

5. Part 1: Preparation of 5 M of NaCl Show calculations involved in lab notebook Draw diagram of how solution is prepared in notebook Store finished mixture at 4 degrees Celsius until ready for use

6. Part 2: Preparation of TE Buffer • Show calculations for TRIS and EDTA mass in lab notebook • Draw a diagram of how the TE buffer solution is prepared in notebook • For pH adjustment: • Slowly add HCl to lower pH • Slowly add NaOH to raise pH

7. Tips Students who finish early should make extra TE buffer for later use? Analytical balances may be used for TRIS and EDTA measurements 5 M NaClis very concentrated; it may take a long time for the sodium chloride to fully dissolve.

8. Lab 4B Pulling DNA out of Solution: DNA Spooling

9. Overview • DNA can be pulled out of aqueous solutions due to certain molecular characteristics: • Long double helix shape • Charged phosphate groups • Repelled by nonpolar solutions, such as ethanol

10. Tips • Do calculations in advance—sperm solution concentration is: • Use the C1V1 = C2V2 equation to determine correct solutions • Record calculations and data in lab notebook • Prepare data table in advance • Keep samples on ice • Ethanol is flammable; keep away from open flame. • We will finish step 12 on Tuesday • When adding ethanol to the DNA/salt mixture, it is very important to have two distinct layers. • Trickle ethanol down the side of the beaker

11. Lab 4C Testing for the Presence of DNA, RNA, and Protein in DNA Extracts

12. DPA Solution • Diphenylamine (DPA) solution is used as an indictor for DNA • Turns blue when DNA is present • Turns green when RNA is present • Indicates that the DNA sample is contaminated • Turns brown or blue-green if both DNA and RNA are present

13. Biuret Indicator • Tests for the presence of protein • Positive test results range from medium blue to violet • Indicates that the DNA sample has yet to be completely purified

14. Tips DPA produces toxic fumes and should only be used in a chemical fume hood with a fan running Poor spooling technique may contaminate DNA sample with alcohol, which will yield inaccurate results

15. Lab 4D EtBR Dot Test: A Quick Test for DNA in Samples

16. Overview • EtBr (ethidium bromide) indicates the presence of DNA by intercalating between nitrogenous base pairs • Causes the bond angles to change, producing different light-absorbing patterns • UV light that shines on an EtBr and DNA mixture will glow pink-organe • Intensity of glow indicates the presence and amount of DNA

17. Tips • Ethanol contamination of samples can result in poor EtBR dot test results • EtBr is known to change the shape of DNA • Therefore, it is a suspected mutagen and carcinogen • Wear goggles and gloves when in near proximity to EtBr